NF-kB signaling lies at the center of Immunodeficiency and Inflammatory diseases. A key regulator of NF-kB signal transduction, NEMO, has recently been found to be mutated in an Immunodeficiency Disorder called called Anhidrotic (hypohydrotic) Ectodermal Dysplasia with Immunodeficiency (EDA-ID). Patients with EDA-ID are susceptible to infection with gram-positive organisms, and molecularly, a dysfunctional NF-kB signaling pathway, owing to hypomorphic mutations in nemo is to blame. While the NF-kB pathway is so well-studied that it has become a paradigm for inflammatory signal transduction, this paradigm has shifted in recent years with the recognition that this pathways signal transduction strength and coordination is critically dependent on Lysine-63 (K63)-linked polyubiquitination. NEMO was very recently shown to contain a ubiquitin binding domain that is essential for NF:B signaling, and NEMO, itself, is K63-polyubiquitinated on two sites in response to innate immune stimulation (NOD2 (Crohn's Disease-susceptibility protein) and Toll-like Receptor (TLR) activation). This NEMO ubiquitination is necessary for optimal NF:B signaling (1, 2, 6, 13, 26). The spectrum and location of point mutations of nemo in EDA-ID suggest that biochemically, they may be interfering with ubiquitin binding by NEMO and/or K63-linked polyubiquitination of NEMO in response to innate immune stimuli. In addition, the varied immunologic phenotypes and immunodeficiencies in EDA-ID suggest cell-type specificity in regards to the particular mutations. Lastly, it has been difficult for the field to uncouple ubiquitin binding by NEMO from K63-linked polyubiquitination of NEMO as it relates to function and ultimately, NF:B-induced gene expression. All these difficulties point to the need to systematically evaluate EDA-ID- associated NEMO mutations in regards to ubiquitin binding, K63-linked polyubiquitination of NEMO and NF-kB-associated gene expression. These difficulties also point to a need for an in vivo model of defective NEMO ubiquitination to decipher its role in in vivo NF:B-dependent gene expression and inflammatory and immunodeficiency disorders. The grant application aims to answer these key questions. PUBLIC HEALTH RELEVANCE: The ability to determine the genetics of susceptibility to infectious disease has led to the identification of novel forms of immunodeficiency, and the challenge is to translate these genetic findings into biochemical mechanisms of disease to both better understand the disease and allow better treatment for that disease. NEMO, a protein that is central to the major inflammatory signaling pathway is mutated in a disease called EDA-ID, a disease that is characterized by susceptibility to bacterial infections. This grant aims to determine how NEMO is faulty in EDA-ID and to generate a mouse model that might mimic many of the features of EDA-ID in hopes that this might lead to a better understanding of and better treatments for this disease.